Fluorescent dyes or stains can be used in the detection of nucleic acids, such as DNA and RNA, present in biological samples. Nucleic acid is the genetic information transmitted from one generation to the next and the routine functioning of a living organism. Nucleic acids are thus of great interest for research and development of diagnostics, therapeutics, forensic tools, and many other applications. Fluorescent dyes that specifically bind to nucleic acids and form highly fluorescent complexes are useful tools for studying nucleic acids. These dyes can be used to detect and quantify DNA and RNA in a variety of environments, including solutions, cell extracts, electrophoretic gels, micro-array chips, live or fixed cells, dead cells, and environmental samples. DNA binding dyes have also been used in quantitative real-time polymerase chain reaction or qPCR, a highly sensitive and specific gene detection technique widely used in both research and diagnostics.
Although numerous fluorescent nucleic acid-binding dyes are commercially available, limitations still exist in various applications. Traditional nucleic acid dyes include the Hoechst dyes, ethidium bromide, DAPI and Thiazole Orange. In general, these dyes have limited sensitivity and high background fluorescence. More recently, higher sensitive nucleic acid dyes have been developed by following two strategies. One strategy is to add positively charged side chains to a dye so that the binding affinity of the dyes is enhanced due to increased electrostatic interaction between the dyes and nucleic acids. Nucleic acid dyes designed using this principle include SYBR Green I, PicoGreen, TO-PRO dyes, YO-PRO dyes and dyes disclosed in U.S. Pat. Nos. 5,436,134; 5,658,751; 4,883,867; 5,582,977; 5,321,130; 5,410,030; and 5,863,753. Another strategy is to link two or three nucleic dyes with a linker to form a dimeric or trimeric dye (see, e.g., U.S. Patent Publication Nos. 2006/0211028 and 2008/0145526). In some cases, the linker may incorporate one or more positive charges to provide additional nucleic acid binding affinity (see, e.g., 5,410,030). Although most of these dyes are only weakly fluorescent in the absence of nucleic acids, the background fluorescence can be significant relative to the fluorescence gain when the amount of target nucleic acid is very small. Consequently, the intrinsic fluorescence of the dye often limits the detection sensitivity of the dye. In real-time PCR, a frequently encountered problem is that the dye inhibits the PCR reaction to some extent. While enough dye must be added to a PCR reaction in order to produce high PCR signal, too much dye can interfere with the PCR process. In some cases, the interference may be severe enough to completely stall the reaction, or generate nonspecific PCR products.